The PDZ Domain of the LIM Protein Enigma Binds to β-Tropomyosin

PDZ and LIM domains are modular protein interaction motifs present in proteins with diverse functions. Enigma is representative of a family of proteins composed of a series of conserved PDZ and LIM domains. The LIM domains of Enigma and its most related family member, Enigma homology protein, bind to protein kinases, whereas the PDZ domains of Enigma and family member actin-associated LIM protein bind to actin filaments. Enigma localizes to actin filaments in fibroblasts via its PDZ domain, and actin-associated LIM protein binds to and colocalizes with the actin-binding protein α-actinin-2 at Z lines in skeletal muscle. We show that Enigma is present at the Z line in skeletal muscle and that the PDZ domain of Enigma binds to a skeletal muscle target, the actin-binding protein tropomyosin (skeletal β-TM). The interaction between Enigma and skeletal β-TM was specific for the PDZ domain of Enigma, was abolished by mutations in the PDZ domain, and required the PDZ-binding consensus sequence (Thr-Ser-Leu) at the extreme carboxyl terminus of skeletal β-TM. Enigma interacted with isoforms of tropomyosin expressed in C2C12 myotubes and formed an immunoprecipitable complex with skeletal β-TM in transfected cells. The association of Enigma with skeletal β-TM suggests a role for Enigma as an adapter protein that directs LIM-binding proteins to actin filaments of muscle cells.

An Atypical Sorting Determinant in the Cytoplasmic Domain of P-Selectin Mediates Endosomal Sorting

We previously identified the 11 amino acid C1 region of the cytoplasmic domain of P-selectin as essential for an endosomal sorting event that confers rapid turnover on P-selectin. The amino acid sequence of this region has no obvious similarity to other known sorting motifs. We have analyzed the sequence requirements for endosomal sorting by measuring the effects of site-specific mutations on the turnover of P-selectin and of the chimeric protein LLP, containing the lumenal and transmembrane domains of the low density lipoprotein receptor and the cytoplasmic domain of P-selectin. Endosomal sorting activity was remarkably tolerant of alanine substitutions within the C1 region. The activity was eliminated by alanine substitution of only one amino acid residue, leucine 768, where substitution with several other large side chains, hydrophobic and polar, maintained the sorting activity. The results indicate that the endosomal sorting determinant is not structurally related to previously reported sorting determinants. Rather, the results suggest that the structure of the sorting determinant is dependent on the tertiary structure of the cytoplasmic domain.

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

The protein p21Cip1, Waf1, Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase δ. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.

The PH Domain and the Polybasic c Domain of Cytohesin-1 Cooperate specifically in Plasma Membrane Association and Cellular Function

Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in ∼100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2–3 μM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the β-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation–isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton’s tyrosine kinase with the adjacent Bruton’s tyrosine kinase motif, a novel zinc-containing fold.

Regulation of Adherence and Virulence by the Entamoeba histolytica Lectin Cytoplasmic Domain, Which Contains a β2 Integrin Motif

Killing of human cells by the parasite Entamoeba histolytica requires adherence via an amebic cell surface lectin. Lectin activity in the parasite is regulated by inside-out signaling. The lectin cytoplasmic domain has sequence identity with a region of the β2 integrin cytoplasmic tail implicated in regulation of integrin-mediated adhesion. Intracellular expression of a fusion protein containing the cytoplasmic domain of the lectin has a dominant negative effect on extracellular lectin-mediated cell adherence. Mutation of the integrin-like sequence abrogates the dominant negative effect. Amebae expressing the dominant negative mutant are less virulent in an animal model of amebiasis. These results suggest that inside-out signaling via the lectin cytoplasmic domain may control the extracellular adhesive activity of the amebic lectin and provide in vivo demonstration of the lectin’s role in virulence.

Efficient Trafficking of TGN38 from the Endosome to the trans-Golgi Network Requires a Free Hydroxyl Group at Position 331 in the Cytosolic Domain

TGN38 is one of the few known resident integral membrane proteins of the trans-Golgi network (TGN). Since it cycles constitutively between the TGN and the plasma membrane, TGN38 is ideally suited as a model protein for the identification of post-Golgi trafficking motifs. Several studies, employing chimeric constructs to detect such motifs within the cytosolic domain of TGN38, have identified the sequence 333YQRL336 as an autonomous signal capable of localizing reporter proteins to the TGN. In addition, one group has found that an upstream serine residue, S331, may also play a role in TGN38 localization. However, the nature and degree of participation of S331 in the localization of TGN38 remain uncertain, and the effect has been studied in chimeric constructs only. Here we investigate the role of S331 in the context of full-length TGN38. Mutations that abolish the hydroxyl moiety at position 331 (A, D, and E) lead to missorting of endocytosed TGN38 to the lysosome. Conversely, mutation of S331 to T has little effect on the endocytic trafficking of TGN38. Together, these findings indicate that the S331 hydroxyl group has a direct or indirect effect on the ability of the cytosolic tail of TGN38 to interact with trafficking and/or sorting machinery at the level of the early endosome. In addition, mutation of S331 to either A or D results in increased levels of TGN38 at the cell surface. The results confirm that S331 plays a critical role in the intracellular trafficking of TGN38 and further reveal that TGN38 undergoes a signal-mediated trafficking step at the level of the endosome.

Mutations in Drosophila Enabled and Rescue by Human Vasodilator-stimulated Phosphoprotein (VASP) Indicate Important Functional Roles for Ena/VASP Homology Domain 1 (EVH1) and EVH2 Domains

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.

Protein Kinase Activity and Identification of a Toxic Effector Domain of the Target of Rapamycin TOR Proteins in Yeast

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.

The Role of the Membrane-spanning Domain Sequence in Glycoprotein-mediated Membrane Fusion

The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.

A Role for the GSG Domain in Localizing Sam68 to Novel Nuclear Structures in Cancer Cell Lines

The GSG (GRP33, Sam68, GLD-1) domain is a protein module found in an expanding family of RNA-binding proteins. The numerous missense mutations identified genetically in the GSG domain support its physiological role. Although the exact function of the GSG domain is not known, it has been shown to be required for RNA binding and oligomerization. Here it is shown that the Sam68 GSG domain plays a role in protein localization. We show that Sam68 concentrates into novel nuclear structures that are predominantly found in transformed cells. These Sam68 nuclear bodies (SNBs) are distinct from coiled bodies, gems, and promyelocytic nuclear bodies. Electron microscopic studies show that SNBs are distinct structures that are enriched in phosphorus and nitrogen, indicating the presence of nucleic acids. A GFP-Sam68 fusion protein had a similar localization as endogenous Sam68 in HeLa cells, diffusely nuclear with two to five SNBs. Two other GSG proteins, the Sam68-like mammalian proteins SLM-1 and SLM-2, colocalized with endogenous Sam68 in SNBs. Different GSG domain missense mutations were investigated for Sam68 protein localization. Six separate classes of cellular patterns were obtained, including exclusive SNB localization and association with microtubules. These findings demonstrate that the GSG domain is involved in protein localization and define a new compartment for Sam68, SLM-1, and SLM-2 in cancer cell lines.

Use of a β1 Integrin-deficient Human T Cell to Identify β1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in β1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the β1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human β1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the β1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the β1 integrin with the activating β1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the β1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of β1 integrin structure and function in human T cells.

Requirement of Sequences outside the Conserved Kinase Domain of Fission Yeast Rad3p for Checkpoint Control

The fission yeast Rad3p checkpoint protein is a member of the phosphatidylinositol 3-kinase-related family of protein kinases, which includes human ATMp. Mutation of the ATM gene is responsible for the disease ataxia-telangiectasia. The kinase domain of Rad3p has previously been shown to be essential for function. Here, we show that although this domain is necessary, it is not sufficient, because the isolated kinase domain does not have kinase activity in vitro and cannot complement a rad3 deletion strain. Using dominant negative alleles of rad3, we have identified two sites N-terminal to the conserved kinase domain that are essential for Rad3p function. One of these sites is the putative leucine zipper, which is conserved in other phosphatidylinositol 3-kinase-related family members. The other is a novel motif, which may also mediate Rad3p protein–protein interactions.

Apoptosis Induced by the Nuclear Death Domain Protein p84N5 Is Inhibited by Association with Rb Protein

Rb protein inhibits both cell cycle progression and apoptosis. Interaction of specific cellular proteins, including E2F1, with Rb C-terminal domains mediates cell cycle regulation. In contrast, the nuclear N5 protein associates with an Rb N-terminal domain with unknown function. The N5 protein contains a region of sequence similarity to the death domain of proteins involved in apoptotic signaling. We demonstrate here that forced N5 expression potently induces apoptosis in several tumor cell lines. Mutation of conserved residues within the death domain homology compromise N5-induced apoptosis, suggesting that it is required for normal function. Endogenous N5 protein is specifically altered in apoptotic cells treated with ionizing radiation. Furthermore, dominant interfering death domain mutants compromise cellular responses to ionizing radiation. Finally, physical association with Rb protein inhibits N5-induced apoptosis. We propose that N5 protein plays a role in the regulation of apoptosis and that Rb directly coordinates cell proliferation and apoptosis by binding specific proteins involved in each process through distinct protein binding domains.

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using β-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain.